Xanthine Oxidase and Xanthine Dehydrogenase from an Estivating

Xanthine Oxidase and Xanthine Dehydrogenase from an Estivating Land Snail Marcelo Hermes-Lima* and Kenneth B. Storey Institute of Biochemistry and Department of Biology, Carleton University, Ottawa, Ontario, Canada K1S 5B6 Z. Naturforsch. 50c, 68 5 -6 9 4 (1995); received January ll/Ju ly 10, 1995 Metabolic Depression, Estivation. Hydrogen Peroxide, Catalase, Oxidative Stress, Gastropod Mollusc, Otala lactea During arousal from estivation in land snails. Otala lactea, active metabolic functions are restored within minutes and oxygen consumption increases dramatically. During the transi tion from the hypoxic conditions of estivation to normoxia it is possible that xanthine oxidase (X O ) in hepatopancreas contributes to the observed lipid peroxidation. Using a fluorometric assay that is based on the oxidation of pterin, the activities and som e properties of XO and X O +X D H (sum of XO and xanthine dehydrogenase activities) were measured in hepato pancreas extracts. Km values for pterin for XO and X O +X D H were 9 and 6 |.im, respectively, and the Km of X D H for methylene blue was 5 |i m . Both X O +X D H and XO activities were inhibited by allopurinol (/50 = 2 j.i m ) , pre-incubation at 40 °C, and by 5 min H20 2 pre-expo sure. Inclusion of azide in the reaction promoted a rise of approximately 70-fold in the inactivation power of H20 2 due to inhibition of high endogenous catalase activity. The /so for H20 2 of X O +X D H and X O activities in the presence o f azide was 0.04 and 0.11 m M , respectively. Unlike the situation for mammalian XO. a previous reduction of O. lactea XO (by pterin) was not necessary to make the enzyme susceptible to H20 2 effects. Interestingly, methylene blue partially prevented both heat- and H20 2-induced inactivation of X O +X D H activity. These data indicate that the formation of an enzym e-m ethylene blue complex in duces protection against heat and oxidative damage at the FAD-active site. Both XO and X O +X D H activities were significantly higher in snails after 35 days o f estivation compared with active snails 24 h after arousal from dormancy. The ratio o f X O /(X O + X D H ) activities was also slightly increased in estivating O. lactea (from 0.07 to 0.09; P < 0.025). XO activity was 0.03 nm ol-m in- 1 -mg protein-1 in estivating snails. Compared with hepatopancreas cata lase, XO activity is probably too low to contribute significantly to the net generation of oxyradicals, and hence to peroxidative damage. Rather, the low potential of XO to induce oxidative stress may constitute an adaptive advantage for O. lactea during arousal periods.

Introduction

Many species of land snails, including Otala lac tea which is native to the area around the M editer ranean sea, enter a state of reduced metabolism known as estivation in order to endure the dry months of the year (Schmidt-Neilsen et al., 1971; B arnhart and McMahon, 1987). During estivation water loss and heart rate are reduced, oxygen con sumption falls to about 30% of the resting value while active, and activities of key enzymes of inter

Abbreviations: EDTA, ethylenediaminetetra-acetic acid; 0 2~, superoxide radical; TBARS, thiobarbituric acid reactive substances; X D H ; xanthine dehydrogenase; XO. xanthine oxidase; IXPT, isoxanthopterin. * Present address: Laboratorio de Biofisica, Departamento de Biologia Celular, Universidade de Brasilia, Brasilia 70910-900, Brasil. Reprint requests to Dr. K. B. Storey. Telefax: 613-788-4389. 0939-5075/95/0900-0685 $ 06.00

mediary metabolism are reduced by means of reversible phosphorylation (e.g. glycogen phosphorylase, phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase) (Herreid, 1977; Whitwam and Storey, 1990, 1991; Brooks and Storey, 1990, 1992; Storey, 1993). Estivation can be rapidly broken by the reintroduction of water into the environment. Snails arouse within minutes, showing an abrupt increase in the rate of oxygen consumption and a quick recovery of active m eta bolic functions (Herreid, 1977; Whitwam and Storey, 1990). We have recently observed that the activities of several antioxidant enzymes in snails were significantly increased after 30 days of estiva tion (Hermes-Lima and Storey, 1995); included are glutathione peroxidase in hepatopancreas, catalase in foot muscle, and superoxide dismutase in both organs. In addition, lipid peroxidation increased significantly in hepatopancreas at the onset of arousal from 30 days of estivation (Hermes-Lima

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68 6

M. H erm es-L im a and K. B. Storey • X anthine O xidase and M etabolie D epression

and Storey, 1995). These results suggested that during estivation snails undergo adaptive changes that will prepare them to deal with a “physiologi cal oxidative stress" (see Barja de Quiroga, 1992) occurring during the arousal process (HermesLima and Storey, 1995). The source of the oxygen radicals that prom ote lipoperoxidation in arousing snails remains un clear. A putative site for the formation of oxygen radicals is the molybdenum-dependent enzyme in volved in purine catabolism, xanthine oxidase (XO). Xanthine oxidase generates superoxide rad icals ( 0 2~) and H 20 2 during the oxidation of xan thine or hypoxanthine (McCord, 1985; Parks and Granger, 1986; Traystman et al., 1991; G reene and Paller, 1992). It has been dem onstrated that XO plays an im portant role in post-hypoxic injury to mammalian tissues (Patt et al., 1988; G reene and Paller, 1992; Terada et al., 1992; Rangan and Bulkley, 1993). Since purine metabolism is a key part of nitrogen excretion in O. lactea (Lee and Cam p bell, 1965; Speeg and Campbell, 1968), we sus pected that XO and xanthine dehydrogenase (XD H) would be active in arousing animals. In fact, the presence of XDH or XO activities has been reported for most prosobranch, pulm onate and bivalve molluscs (Krenitsky et al., 1974; Bishop et al., 1983; Dykens and Shick, 1988; Ramesh et al., 1990). Moreover, taking into con sideration that the process of arousal is a transi tion from hypoxia (PQ, is low in tissues during estivation; Barnhart, 1986a) to normoxia, the pres ence of XO (and/or conversion of XDH into XO) could set up a potential risk of oxidative damage. Dykens and Shick (1988) have proposed that XO could exert oxidative stress in marine bivalves over the course of the tidal cycle. According to their proposal, molluscs presenting poor tolerance to anoxia show conversion of XDH into XO and notable adenylate degradation causing accumu lation of XO substrates (xanthine and hypo xanthine) during aerial exposure. These animals would then be susceptible to oxygen reperfusion injury during reimmersion at high tides. The present study analyzes the kinetic proper ties of XO and XDH from O. lactea hepatopancreas using pterin as a substrate, which allows for a very sensitive fluorometric quantification of the activities (Beckman et al., 1989). The effects of H20 2 on both XO and XDH activities were ana

lyzed since H 20 2 is a product of the XO-catalyzed reaction and could induce XO self-inactivation (Terada et al., 1991), as well as XDH damage. Maximal activities of XO and X D H were also quantified in snails after 35 days of estivation ver sus 24 h after arousal. Materials and M ethods

Chemicals Allopurinol, bovine milk xanthine oxidase, EDTA. isoxanthopterin, pterin, methylene blue, phenylmethylsulfonyl fluoride, and Sephadex G-25 were purchased from Sigma Chemical Co. All other reagents were of analytical grade. Animals Land snails O. lactea were obtained from a com mercial supplier in Ottawa. Canada. Animals were held in the laboratory at 22 °C in covered plastic containers lined with paper towels. Every 30-40 days animals were sprayed with dechlorinated water to induce arousal, then fed cabbage sprin kled with ground chalk; animals were then allowed to reenter dormancy over the next several days. For experimental sampling, dorm ant snails were sampled after 35 days of continuous dormancy as described above. At the same time another group of animals was sprayed with water, aroused and fed. A fter 24 h in the active state, this group of aroused snails was sampled. For sampling purposes, snails were killed by breaking the shell and organs (foot and hepatopancreas) were quickly dissected out, frozen in liquid nitrogen, and then transferred to -7 5 °C for storage. Preparation o f tissue extracts for enzym e assays Frozen tissue samples were quickly weighed and immediately homogenized (1:10 w/v) in cold 50 mM potassium phosphate buffer, pH 7.0 con taining 0.5 mM EDTA and with a few added crys tals of phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 5 °C and 25,000xg for 25 min. Supernatants were removed and 0.5 ml aliquots were filtered through 5 ml columns of Sephadex G-25 (with centrifugation for 1 min in a benchtop centrifuge (Helmerhorst and Stokes, 1980) to remove endogenous low molecular weight inhibitors. Extracts were then stored on ice


and used for experiments. Hepatopancreas XDH and XO activities were 4 0 -60% and almost com pletely inhibited, respectively, in supernatants that were not desalted by G-25 filtration. Initial attempts were made to partially purify XO and XO+XDH using DE-52 ion exchange chromatographys (elution with a salt gradient). Both en zymes eluted in single peaks but activities were unstable, with a 50% loss of activity in only 1 day at 4 °C; hence, these were unsuitable for use. Measurement o f the activities o f X O and X D H Activities were followed by the fluorometric assay of Beckman et al. (1989) based on the XO/ XDH-catalyzed conversion of pterin to isoxan thopterin. Assays were m onitored with 345 nm ex citation and 390 nm emission wavelength on a Per kin Elm er LS-50 fluorometer. In the standard assay procedure, 50 [a 1 of enzyme extract was added to 50 mM potassium phosphate (pH 7.0) buffer containing 0.5 mM EDTA (at 25 °C). Then 10 [il of 2 m M pterin was added to record the XO activity. A fter 3 - 4 minutes, 10 |a 1 of 1 mM m ethy lene blue was added in order to follow the sum of XO and XDH activities (XO+XDH activity). Methylene blue was used to replace N A D + as the final electron acceptor for XDH. A fter recording the XO+XDH activity the reaction was stopped by addition of 10 [Al of 4 m M allopurinol, followed by addition of 2 (il of 0.02 m M isoxanthopterin (final volume of reaction: 1.01 ml) in order to cali brate the fluorometric assay. In order to control for accidental conversion of XDH to XO during homogenization due to sulfhydryl redox changes we tested the effect of dithiothreitol (5 mM) addition. There were no sig nificant differences in either XO or XO+X D H activities from samples (from hepatopancreas of estivating snails) prepared with or without the ad dition of dithiothreitol in the homogenization buffer. These results indicated that the measured XO activity in homogenates represented an active XO in O. lactea hepatopancreas in vivo. Analysis o f data All results were computed as mean ± S.E. of 2 - 4 independent experiments using different tis sue extracts. One-tailed Student's t-tests were em

687

ployed to analyze the data. The level of statistical significance was taken as P < 0.05. Results

Hepatopancreas of O. lactea showed both XO and X O +X D H activities. Figure 1 shows the de pendence of hepatopancreas XO activity on pterin concentration (using enzyme from 24 h aroused snails). A ddition of 10 [a m methylene blue after 3 - 4 min of monitoring XO activity allowed the further m easurem ent of the XO+XDH activity. The apparent K m values for pterin were 6.0 ± 0.9 |a m and 8.6 ± 5.1 [a m for the XO+XDH and XO activities, respectively, similar to K m values for mammalian XO using xanthine as substrate (2 -8 [a m for bovine milk or mouse, human and rat liver) (Waud and Rajagopalan, 1976; Krenitsky et al., 1986; Carpani et al., 1990). In addition, a typical Michaelis-Menten curve for XDH was obtained with increasing concentrations of methylene blue; a K m of 5 [a m was obtained (Fig. 2). The pH dependence of the XO+XDH activity showed a typical bell-shaped profile, and maximal activity was observed at pH 7.8 (Fig. 3). In the case of XO, a similar result was obtained, although a broader pH range (7.0-7.8) presented optimal ac tivity. Figure 4 shows the effect of the XO inhibi-

[Pterin] (//M) Fig. 1. D ependence of O. lactea hepatopancreas XO and X O +X D H activity on pterin concentration. Concentra tion of m ethylene blue in the assay for X O +X D H was 10 [a m . Enzymatic activity was followed as described in Material and Methods. Values are the mean ± S.E. with n = 3 for X O +X D H and n = 2 - 3 for XO.

M. H erm es-L im a and K. B. Storey • X an th in e O xidase and M etabolie D epression

688

[A llo p u r in o l] (//M)

Methylene Blue (fiU) Fig. 2. Dependence of X O +X D H activity on methylene blue concentration. Concentration of pterin in the assay was 20 pM. Inset shows a Lineweaver-Burk plot o f X D H activity (X D H values calculated as X O +X D H values XO values). Values are the mean ± S.E. with n = 3.

PH Fig. 3. Effect of pH on XO and X O +X D H activities. Buffers used were citric acid/potassium phosphate 25mM:25mM (pH 5.5-6.5): 50 mM potassium phosphate (pH 7.0 and 7.8) and 50 mM Tris-HCl (pH 8.4 and 9.0). Values are the mean ± S.E. (n = 3 except for pH 8.4 and 9). *: Fluorescence intensity was corrected relative to pH 7.0 values.

tor, allopurinol (Parks and Granger, 1986), on the activities of hepatopancreas XO+XDH and XO. An / 50 value of 2 [.i m was found in both cases, which is similar to values for human liver XO (Krenitsky et al., 1986). It is proposed that allo purinol inhibits XO activity due XO-induced con-

Fig. 4. Effect o f allopurinol concentration on X O +X D H activity. The inset shows the inhibitory effect on XO ac tivity alone. Allopurinol up to 0.1 m M caused no fluores cence interference. Values are the mean ± S.E. with

n - 3.

version of allopurinol to alloxanthine (oxypurinol), which interacts with the molybdenum site of the enzyme with very high affinity (Parks and Granger, 1986). The time course of heat (40 °C) inactivation of X O +X D H activity (Fig. 5A) exhibited a typical first order profile consisting of two-phases (Fig. 5A inset). A fter 15 min at 40 °C only about 35% of the original enzyme activities were still present. A similar result was observed for XO activity alone. Incubation of extracts at 26 °C induced a slower loss of X O+XDH activity (half-time of about 40 min) (Fig. 5A inset). Addition of 10 |.i m m ethylene blue to the reaction media during a 15 min heat (40 °C) exposure lead to about 54% protection of XO+XDH activity (Fig. 5B). This in dicates that the formation of the enzyme-methylene blue complex stabilizes the enzyme. Addition of hydrogen peroxide resulted in an inactivation of O. lactea XO+XDH activity in crude extracts (reaction media containing 50 ^1 ex tract pre-incubated for 5 min in the presence of H 20 2 before addition of substrates). Under these conditions the / 50 for H 20 2 was 2.7 ± 0.6 mM (Fig. 6). However the presence of a high activity of catalase in hepatopancreas extracts (170-180 U/mg protein; Hermes-Lima and Storey, 1995) could be responsible for an underestimate of the / 50 value for H 20 2. Thus, the addition of 2 mM azide, which fully inhibits catalase activity, con siderably enhanced the inhibitory potential of


689

B.

A.

Fig. 5. (A ) Time course of heat inactivation of X O + X D H and XO activities. Hom ogenates were pre-incubated at 40 °C for different times and 50 ^1 aliquots were removed for activity measurements at room temperature. The inset shows a log scale o f the heat inactivation of X O +X D H at 40 °C or 26 °C. (B ) Protective effect of methylene blue (M B ) against 15 min heat exposure at 40 °C. M eth ylene blue was added at a concentration o f 0.2 mM to the homogenates. and upon addition of hom oge nates to the reaction mixture methylene blue was diluted to 10 |i m . Values are the mean ± S.E. with n = 2 - 3 . The percentage protection provided by m ethylene blue was calculated from the formula: 100 x (heat plus MB) - (heat) / (control) - (heat).

P r e -in c u b a t io n at 40 C (m in)

H 20 2 for the XO+XDH activity, and an actual h o value could be measured (0.04 ± 0.01 m M ). XO activity (in the presence of azide) was also highly susceptible to H 20 2-induced damage (I5Q about 0.1 mM; Fig. 6 inset). We have recently observed a protective effect of endogenous catalase on the OH-induced inactivation of glutathione S-transferase in extracts of garter snake muscle (HermesLima and Storey, 1993a). Figure 7 depicts the time dependence of XO+XDH inactivation due to

0.2 m M H 20 2 in media containing azide. Second order kinetics were observed for the inactivation (inset to Fig. 7), with a calculated t 1/2 of 1.2 ± 0.1 min. Interestingly, 10 jam methylene blue also pro tected X O +X D H from inactivation promoted by 0.2 mM H 20 2 (Table I), although pterin conferred no protection. If methylene blue was causing pro tection by forming an E - S complex, it would be expected that lower levels of methylene blue

0

2

4

6

8

10

Time (m in) 1

2

3

4

8

[H20 2] (mM) Fig. 6. D ependence of X O +X D H activity on H20 2 con centration in reaction media in the absence or presence of 2 mM azide. Reaction media were incubated 5 min in the presence of H 20 2 prior to addition yof the enzyme substrates. Inset shows the effect of H20 2 on the activity of XO alone in media containing 2 mM azide.

Fig. 7. Time course of H 20 2-induced loss of X O +X D H activity. Incubations containing 50 mM potassium phos phate, 0.5 m M E D T A , 2 m M azide and 50 j_il of hom ogenate were pre-exposed to 0.2 mM H20 2 prior to addition of pterin (20 |i m ) and m ethylene blue (10 ^.m ) (filled cir cles). Open circles show incubations without pre-expo sure to H 20 2. The inset shows a second order kinetics profile of X O + X D H inactivation. The straight line in the graph is a computer generated linear regression.

690

M. H erm es-L im a an d K. B. Storey • X an th in e O xidase and M etabolie D epression

Table I. Protective effect of m ethylene blue (M B) against 0.2 mM H20 2-induced X O +X D H inactivation. [MB] 2.5

X O + X D H activity (fluorescence intensity/min) with MB pre-incubationa without M B1’

(.im

- H , 0 2 2.69 ± 0.12 + H20 2 1.33 ± 0.15 (51.3 ± 6.0%)

3.05 ± 0.42 0.57 ± 0.03 (19.1 ± 1.7%)

10 (AM - H . 0 2 4.84 ± 0.55 3.18 ± 0.96 + H 20 2 3.18 ± 0.96 (65.4 ± 4.3% )c 0.79 ± 0.11 (20.6 ± 3.8%)

a Buffered reaction mixtures containing 50 |j,l of tissue extract (with or without 0.2 mM H20 2) were pre-incubated in the presence of 2.5 or 10 [am methylene blue for 5 min prior to the addition of pterin. b Reaction mixtures were pre-incubated for 5 min (with or without 0.2 mM H 20 2) prior to addition of both pterin and methylene blue. Results are means ± S.E.M. (n = 3 - 6 different extracts) with units in rel ative fluorescence intensity/min. Values in brackets show the percentage of activity remaining compared with corresponding controls without H20 2 addition. c The percentage of activity remaining is significantly different from the value for incubation with 2.5 |.im m ethylene blue, P < 0.05, paired t-test.

XO+XDH

XO

Fig. 8. A ctivities of X O +X D H and XO (nmol isoxan thopterin produced-m in mg protein *) in hepato pancreas of snails after 35 days of estivation (filled bars) and 24 h after arousal (open bars). Inset shows the per centage o f total X O +X D H that was XO in dormant and active O. lactea hepatopancreas.

Discussion

would lead to less protection. Indeed, 2.5 [.i m m eth ylene blue induced less protection than did 10 [im m ethylene blue (P < 0.05). Control experiments dem onstrated that methylene blue was not de stroying H 20 2 in solution because pre-incubation of H 20 2 with methylene blue for 1 h produced the same level of XO+XDH activity as did samples with no pre-incubation. Thus, these data suggested that H 20 2 was inducing damage at the FAD-containing active site of XDH. where m ethylene blue is reduced. Finally, Fig. 8 shows the activities of XO +X D H and XO (expressed per mg protein) in hepatopancreas of snails following 35 days of dormancy and 24 h of arousal. Both X O+XDH and XO activities were 2.3- and 3.0-fold higher, respec tively, in hepatopancreas of dorm ant, com pared with active snails. In addition, the percentage of total activity that was XO was also higher in the dorm ant snails, 9.6 ± 0.7 % compared with 7.0 ± 0.4 % in the snails 24 h after arousal. Very low levels of XDH, with no detectable XO activity, were found in foot muscle (30-40 times lower than activities in hepatopancreas) and the en zymatic activity was allopurinol sensitive (data not shown).

Xanthine oxidase and/or XDH participate in the catabolic pathway for the breakdown of purine nucleotides to xanthine and uric acid in most ani mal species (Parks and Granger. 1986). They also play an im portant role in post-hypoxic oxidative injury in various mammalian organs (Beckman et al., 1986; Rangan and Bulkley, 1993). About 90% of the nitrogen in the excreta of pulmonate land snails is in the form of guanine, uric acid, and xanthine (Bishop et al., 1983). This correlates with the fact that m easurable levels of both XO and XDH were found in O. lactea hepatopancreas. The observed increase in XO and XO+XDH activities during estivation (Fig. 8) is a possible response to the need to produce relatively non-toxic nitrogen excretion products that can be safely accumulated in the body over long periods of dormancy. Xan thine and uric acid accumulates in the kidneys of estivating O. lactea at rates of 15-25 and 55-90 nm ol-g whole body-1 day-1, respectively (Speeg and Campbell, 1968). The allopurinol-sensitive XO +X D H activity in hepatopancreas of 35 day dorm ant snails was about 0.3 nmol isoxantho pterin m in^1-mg protein-1 (12 m U -g wet w t-1). This pterin oxidation activity is about the same as that reported for human liver, although 6 times lower than that in rat liver (activities at 25 °C and


reported per mg protein) (Beckman et al., 1989). Based on the com parative measurements of Beck man et al. (1989) for mammalian X O+XDH activi ties, we could predict that rates 2 - 4 fold higher (compared with pterin) would be expected for the oxidation of xanthine catalyzed by O. lactea X O+XDH. This expected activity of O. lactea X O+X D H is com parable to that reported in Mytilus edulis, a marine mussel that also undergoes m etabolic arrest (in response to anoxia); however, this species displays only XDH (Dykens and Shick, 1988; Dykens et al., 1989). The pH optimum of X O+XDH was 7.8 (Fig. 3) but about 80 % of maximal activity was retained at pH 7. By contrast, the pH optimum of bovine milk XO (pterin as substrate) was 5.5 (Beckman et al., 1989). Intracellular pH in O. lactea is about pH 7.5 in active animals and undoubtedly falls during estivation; hemolymph pH fell by about 0.4 units after one m onth of estivation (Barnhart, 1986b) and Barnhart and McMahon (1988) showed that intracellular pH paralleled extracellu lar pH over a wide range of P c o values. Hence, pH effects may tend to reduce enzyme activity in estivating animals although other factors, such as elevated substrate concentrations in estivating snails may be more influential on enzyme activity in vivo. The increase in the X O /(X O+XDH ) activity ratio in estivation (Inset to Fig. 8) may be ex plained by a X D H to XO conversion via the action of proteases. It has been proposed that Ca2+-dependent proteases catalyze an irreversible conver sion of X D H to XO in mammals (McCord, 1985; G ranger et al., 1986). Interestingly, a rise in pro tease activities during estivation has been reported in another snail species (Ramesh et al., 1990). In the case of O. lactea, no change in the amount of total soluble protein (m easured per g wet weight) was detected after one month of estivation (Herm es-Lim a and Storey, 1995). However, this does not exclude the possibility that proteases play a role in the increase in the O. lactea X O / (X O +X D H ) ratio. Reversible oxygen-dependent conversion of X D H to XO during estivation is un likely because of the low PQl in the tissues of esti vating snails (e.g. 0.35 kPa in the lung) (Barn hart, 1986a). Could XO and X D H be linked with an induc tion of oxidative stress in O. lacteal Many studies

691

have dem onstrated the role of XO in the genera tion of oxyradicals in mammalian systems under pathological situations (McCord, 1985; G ranger et al., 1986; Traystman et al., 1991; Rangan and Bulkley, 1993). During ischemia the catabolism of ATP leads to an accumulation of xanthine and hypoxanthine (Rubin et al., 1992) and Ca2+-dependent conversion of XDH into XO (Traystman et al., 1991). Reperfusion with oxygenated blood leads to the formation of 0 2- and H 20 2 (catalyzed by XO or other sources of oxyradical form ation) as well as highly reactive OH radicals (Patt et al., 1988; Jaeschke and Mitchell, 1989). Hydroxyl radi cals induce widespread cellular injuries including mitochondrial damage and lipid peroxidation (Traystman et al., 1991; Hermes-Lima et al., 1991; Henry et al., 1993): H 20 2 + 0 2-

(Fe3+, Cu2+) — 0 2 + O H " + OH.

During dormancy the activity of XO in hepatopancreas increased 3-fold after 35 days of estiva tion (Fig. 8), reaching 0.03 nmol isoxanthopterinm in-1 -mg protein-1 (1.2 m U -g wet weight-1). In addition, the XO substrate (xanthine) accumulates slowly in the kidney of estivating snails (Speeg and Campbell, 1968), and probably also in other tis sues. Upon arousal, oxygen consumption rises d ra matically (Herreid, 1977) and tissue P0 , quickly returns to normoxic values (Barnhart, 1986a). We have previously m easured the levels of thiobarbituric acid reactive substances (TBARS) in hepatopancreas during arousal from estivation; the assay quantifies a series of aldehydic products of peroxi dation including malonaldehyde and 4-hydroxynonenal (Uchiyama and Mihara, 1978; HermesLima and Storey, 1995). A transient increase in TBARS content of 25 % occurred after 20 min of arousal (TBARS rose from 26.3 ± 2.7 to 32.8 ± 1.1 nm ol-g wet w t-1, P < 0.05, n = 5) (HermesLima and Storey, 1995) indicating that damage by oxyradicals increases early in arousal. Moreover, the expected activity of O. lactea XO toward xan thine oxidation (see above) is also comparable with the XO activities of the marine stenoxic bi valves Pecten maximus and Placopecten magellanicus (Dykens and Shick, 1988) and it has been pro posed that XO is implicated in post-anoxic injury in these animals. Furtherm ore, the activity of XO in estivating O. lactea is comparable with rat brain XO activity (accounting for 79% of the total rat

692


brain X O +X D H activity) (Margolin and Behrman, 1992), and is suggested to play a role in cere bral post-ischemic oxidative damage (Beckman et al., 1986; Patt et al., 1988; Kinuta et al., 1989). Taking all these factors as a whole, it could be pos sible that XO contributes to oxyradicals genera tion in O. lactea during arousal. On the other hand, based on the hepatopancreas XO activity using a natural substrate (xanthine), we can predict a rate of H 20 2 genera tion by XO of 0 .6 -1 .2 x l 0 -4 [im o lm in -1 -mg protein-1 (equivalent to 0.03 nmol pterin oxida tion m in“ 1-mg protein-1) (F H,o )- However, even under optimum conditions F H,0 , would be over whelmed by hepatopancreas catalase activity (170-180 |_imol H 20 2 m in-1 • mg protein-1, rela tive to 10 m M substrate; Herm es-Lim a and Storey, 1995). This would also be true considering a cata lase activity of about 2 x l 0 -3 |.imol H 20 2-m in_1. mg protein ( D H ,0 ,) based on a realistic in vivo steady state concentration of H 20 2 of about 10~7 m (Chance et al., 1979). The calculated ratio D H2o 2/ F H2o 2 (~ 0 .2 -0 .3x 102), which still under estimates the role of glutathione peroxidase and other peroxidases in the decomposition of H 20 2, suggests that XO does not play a major role in oxyradicals generation and oxidative damage in O. lactea. Thus, other putative sources of oxyradi cals generation during arousal need to be consid ered including the P450 detoxification system, which has been identified in a variety of molluscs (Livingstone, 1991), and the “electron leak” at the mitochondrial respiratory chain (Jaeschke and Mitchell, 1989; Konstantinov et al., 1987). It is noteworthy that in mammalian systems, such as rat liver or pigeon heart, about 1 -4 % of consumed oxygen is converted to 0 2~ and H 20 2 at the m ito chondrial level (Turrens et al., 1982; Cadenas and Boveris, 1980; Konstantinov et al., 1987), and this could also be true in the case of O. lactea. On the other hand, the correlation between the lack of m easurable XO activity and the lack of change in TBARS levels following arousal in foot muscle of O. lactea (mean TBARS were only 5.7 ± 0.4 nmolg wet w eight-1; Hermes-Lima and Storey, 1995) cannot be ignored. The high susceptibility of O. lactea XO+XDH and XO to H 20 2 effects (/50 in the presence of azide = 0.04 m M ) differs from the case of purified bovine milk XO where only millimolar levels

(5 -3 0 mM) of H20 2 were found to be damaging (Terada et al., 1991). Moreover, the requirem ent of xanthine (which reduces XO) for the H 20 2-induced inactivation of bovine milk XO was not nec essary in the case of O. lactea XO (and XDH as well). Terada et al. (1991) have postulated that damaging OH radicals would be formed at the active site of XO as electron transfer from FA D H 2 to H 20 2 takes place. Therefore, a reduced form of the enzyme is required for the inactivation by H 20 2. This mechanism seems unlikely in the case of O. lactea XO. The decrease in the activity of XO and XO+XDH in snails after 24 h of arousal could be only partially explained by a mechanism of H 20 2induced inactivation. Although endogenous anti oxidant enzymes should protect hepatopancreas XO and XO+XDH activities from H 20 2 effects, it is plausible that mitochondrial generation of oxy radicals would inflict some damage to these en zymes in vivo during arousal. Nevertheless, direct evidence for XO/XD H damage in vivo has yet to be found in O. lactea. The mechanism proposed by Terada et al. (1988) for hyperoxic injury in m am malian lung involving self-inactivation of XO (a potential feedback mechanism for cellular protec tion) seems unlikely in the case of O. lactea due to the low expected rates of XO-induced H 20 2 for mation. Finally, the reduction in hepatopancreas XO and XO+XDH activities could be interpreted as a down regulation process in order to redirect nitrogen metabolism towards pathways that could, for example, supply purines for nucleic acid bio synthesis in active snails (Speeg and Campbell. 1968; Bishop et al., 1983). In conclusion, despite the observed 3-fold increase in the activity of O. lactea hepatopancreas XO in estivating snails (35 days) it appears that XO only plays a secondary role in exerting oxida tive stress because of (i) the low expected F H,Q, values and (ii) the high D H, o values in hepato pancreas. We have previously proposed that the increase in antioxidant enzyme activities in hepa topancreas during estivation (superoxide dismutase and glutathione peroxidase) can be regarded as a mechanism of protection against post-hypoxic stress (Hermes-Lima and Storey, 1995). Both the low activity of hepatopancreas XO and the limited conversion of XDH into XO during estivation (Fig. 8) may also represent an adaptive advantage

M. H erm es-L im a and K. B. S torey • X anthine O xidase and M etabolie D epression

that minimizes oxidative stress following arousal. The lower activity of XO after 24 h arousal would make X O-mediated oxyradicals generation even less significant for active animals. Indeed, these adaptations (regarding X O /XD H and antioxidant enzymes status) may be the reason that only a very limited am ount of lipid damage is observed following arousal (only 25%) (Hermes-Lima and Storey, 1995). Similarly, we have previously re ported that several antioxidant enzyme activities were increased in garter snakes during anoxia or freezing tolerance as a preparation for potential oxygen reperfusion injury following reoxygenation or thawing (Hermes-Lima and Storey, 1993b;

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